1. Field of the Invention
This invention relates to a confocal scanning type of phase contrast microscope for forming a visible image representing the phase information of a transparent sample. This invention also relates to a scanning microscope, which can form an ordinary bright field image and a visible image representing the phase information of a transparent sample.
2. Description of the Prior Art
Optical type scanning microscopes have heretofore been used. With the scanning microscope, a light beam is converged to a small light spot on a sample, and the sample is two-dimensionally scanned with the light spot. The light beam, which has passed through the sample during the scanning, the light beam, which has been reflected from the sample during the scanning, or the fluorescence, which is produced by the sample during the scanning, is detected by a photodetector. An enlarged image of the sample is thereby obtained.
As one type of the scanning microscopes, a confocal scanning microscope has heretofore been proposed. With the confocal scanning microscope, a light beam is produced by a light source and is condensed to a light spot such that an image of the light spot is formed on a sample. Also, a point image of the light beam, which has been radiated out of the sample, is formed and detected by a photodetector. The confocal scanning microscope is advantageous in that no pinhole plate need be located on the surface of the sample.
Basically, the confocal scanning microscope comprises:
i) a light source which produces a light beam, PA0 ii) a sample supporting member on which a sample is supported, PA0 iii) a light projecting optical means with which an image of the light beam is formed as a small light spot on the sample, PA0 iv) a light receiving optical means with which the light beam radiated out of the sample (i.e. the light beam, which has passed through the sample, the light beam, which has been reflected from the sample, or the fluorescence produced by the sample) is condensed, and an image of the condensed light beam is formed as a point image, PA0 v) a photodetector which defects the point image, and PA0 vi) scanning mechanism for two-dimensionally scanning the sample with the light spot. PA0 (1) a mechanism which two-dimensionally moves the sample supporting member, and PA0 (2) a mechanism which two-dimensionally deflects the light beam by a light deflector. PA0 i) a sample supporting member on which a sample is supported, PA0 ii) a light source which produces a light beam, PA0 iii) a light projecting optical means with which an image of said light beam is formed as a small light spot on said sample, PA0 iv) a light receiving optical means with which the light beam radiated out of said sample is condensed, and an image of the condensed light beam is formed as a point image, PA0 v) a photodetector which detects said point image, PA0 vi) a phase difference optical means provided with a phase plate and a stop, one of which is located on the side of said light projecting optical means and the other of which is located on the side of said light receiving optical means, said phase difference optical means imparting a phase lag to only either one of a light component of said light beam, which light component has been diffracted by said sample, and an undiffracted light component of said light beam, which undiffracted light component has passed through said sample or has been reflected by said sample, and thereafter causing said diffracted light component and said undiffracted light component to interfere with each other, PA0 vii) a movable member which supports said light projecting optical means, said light receiving optical means, and said phase difference optical means together, PA0 viii) a main scanning means which reciprocally moves said movable member such that said light spot scans said sample in main scanning directions, and PA0 ix) a sub-scanning means which moves said sample supporting member with respect to said movable member in sub-scanning directions, which are approximately normal to said main scanning directions, and at a speed lower than the speed at which said light spot scans said sample in said main scanning directions, whereby said sample is scanned with said light spot in said sub-scanning directions. PA0 i) a sample supporting member on which a sample is supported, PA0 ii) a light source for producing a light beam including a light component, which has been polarized linearly in a first direction, and a light component, which has been polarized linearly in a second direction that intersects perpendicularly to said first direction, PA0 iii) a first polarizer which has a predetermined shape, said first polarizer transmitting only said light component, which has been polarized linearly in said first direction, therethrough, PA0 iv) a second polarizer for transmitting only said light component, which has been polarized linearly in said second direction, therethrough, PA0 v) a light projecting optical means for forming an image of the light beam composed of said light components, which have respectively passed through said first polarizer and said second polarizer, said image being formed as a small light spot on said sample, PA0 vi) a scanning means which causes said light spot to scan said sample two-dimensionally, PA0 vii) an analyzing phase plate which has a shape corresponding to the shape of said first polarizer, said analyzing phase plate transmitting only said light component, which has been polarized linearly in said first direction, therethrough, said light component having been radiated out of said sample (i.e. having passed through said sample or having been reflected by said sample), a phase lag being thereby imparted to said light component, which has been polarized linearly in said first direction, PA0 viii) a polarization beam splitter which is located on the side downstream from said analyzing phase plate, said polarization beam splitter separating said light component, which has been polarized linearly in said first direction, and said light component, which has been polarized linearly in said second direction, from each other, PA0 ix) a first light receiving optical means for condensing said light component, which has been polarized linearly in said first direction, said light component having been separated by said polarization beam splitter from said light component, which has been polarized linearly in said second direction, an image of the condensed light component being thereby formed as a point image, PA0 x) a second light receiving optical means for condensing said light component, which has been polarized linearly in said second direction, said light component having been separated by said polarization beam splitter from said light component, which has been polarized linearly in said first direction, an image of the condensed light component being thereby formed as a point image, PA0 xi) a first photodetector for detecting said point image, which has been formed by said first light receiving optical means, and PA0 xii) a second photodetector for detecting said point image, which has been formed by said second light receiving optical means. PA0 i) a sample supporting member on which a sample is supported, PA0 ii) a light source for producing a light beam including a light component, which has been polarized linearly in a first direction, and a light component, which has been polarized linearly in a second direction that intersects perpendicularly to said first direction, PA0 iii) a phase plate which has a predetermined shape, said phase plate imparting a phase lag to part of said light beam, which part passes through said phase plate, PA0 iv) a polarizer for transmitting only said light component, which has been polarized linearly in said first direction, therethrough, said light component being included in part of said light beam, which part does not pass through said phase plate, PA0 v) a light projecting optical means for forming an image of the light beam composed of said part of said light beam, which part has passed through said phase plate, and said light component, which has been polarized linearly in said first direction, said light component having passed through said polarizer, said image being formed as a small light spot on said sample, PA0 vi) a scanning means which causes said light spot to scan said sample two-dimensionally, PA0 vii) a first analyzer for transmitting only said light component, which has been polarized linearly in said first direction, therethrough, said light component having been radiated out of said sample, PA0 viii) a second analyzer which has a shape corresponding to the shape of said phase plate, said second analyzer transmitting only said light component, which has been polarized linearly in said second direction, therethrough, said light component being included in said part of said light beam, which part has been imparted with said phase lag and has been radiated out of said sample, PA0 ix) a polarization beam splitter for separating said light component, which has been polarized linearly in said first direction, said light component having passed through said first analyzer, and said light component, which has been polarized linearly in said second direction, said light component having passed through said second analyzer, from each other, PA0 x) a first light receiving optical means for condensing said light component, which has been polarized linearly in said first direction, said light component having been separated by said polarization beam splitter from said light component, which has been polarized linearly in said second direction, an image of the condensed light component being thereby formed as a point image, PA0 xi) a second light receiving optical means for condensing said light component, which has been polarized linearly in said second direction, said light component having been separated by said polarization beam splitter from said light component, which has been polarized linearly in said first direction, an image of the condensed light component being thereby formed as a point image, PA0 xii) a first photodetector for detecting said point image, which has been formed by said first light receiving optical means, and PA0 xiii) a second photodetector for detecting said point image, which has been formed by said second light receiving optical means. PA0 i) a sample supporting member on which a sample is supported, PA0 ii) a light source for producing a light beam including a light component, which has a first wavelength, and a light component, which has a second wavelength different from said first wavelength, PA0 iii) a first filter which has a predetermined shape, said first filter transmitting only said light component, which has said first wavelength, therethrough, PA0 iv) a second filter for transmitting only said light component, which has said second wavelength, therethrough, PA0 v) a light projecting optical means for forming an image of the light beam composed of said light components, which have respectively passed through said first filter and said second filter, said image being formed as a small light spot on said sample, PA0 vi) a scanning means which causes said light spot to scan said sample two-dimensionally, PA0 vii) a wavelength selective phase plate which has a shape corresponding to the shape of said first filter, said wavelength selective phase plate transmitting only said light component, which has said first wavelength, therethrough, said light component having been radiated out of said sample, a phase lag being thereby imparted to said light component, which has said first wavelength, PA0 viii) a light splitting means which is located on the side downstream from said wavelength selective phase plate, said light splitting means separating said light component, which has said first wavelength, and said light component, which has said first wavelength, from each other, PA0 ix) a first light receiving optical means for condensing said light component, which has said first wavelength, said light component having been separated by said light splitting means from said light component, which has said second wavelength, an image of the condensed light component being thereby formed as a point image, PA0 x) a second light receiving optical means for condensing said light component, which has said second wavelength, said light component having been separated by said light splitting means from said light component, which has said first wavelength, an image of the condensed light component being thereby formed as a point image, PA0 xi) a first photodetector for detecting said point image, which has been formed by said first light receiving optical means, and PA0 xii) a second photodetector for detecting said point image, which has been formed by said second light receiving optical means. PA0 i) a sample supporting member on which a sample is supported, PA0 ii) a light source for producing a light beam including a light component, which has a first wavelength, and a light component, which has a second wavelength different from said first wavelength, PA0 iii) a phase plate which has a predetermined shape, said phase plate imparting a phase lag to part of said light beam, which part passes through said phase plate, PA0 iv) a light projecting side filter for transmitting only said light component, which has said first wavelength, therethrough, said light component being included in part of said light beam, which part does not pass through said phase plate, PA0 v) a light projecting optical means for forming an image of the light beam composed of said part of said light beam, which part has passed through said phase plate, and said light component, which has said first wavelength, said light component having passed through said light projecting side filter, said image being formed as a small light spot on said sample, PA0 vi) a scanning means which causes said light spot to scan said sample two-dimensionally, PA0 vii) a first light receiving side filter for transmitting only said light component, which has said first wavelength, therethrough, said light component having been radiated out of said sample, PA0 viii) a second light receiving side filter which has a shape corresponding to the shape of said phase plate, said second light receiving side filter transmitting only said light component, which has said second wavelength, therethrough, said light component being included in said part of said light beam, which part has been imparted with said phase lag and has been radiated out of said sample, PA0 ix) a light splitting means for separating said light component, which has said first wavelength, said light component having passed through said first light receiving side filter, and said light component, which has said second wavelength, said light component having passed through said second light receiving side filter, from each other, PA0 x) a first light receiving optical means for condensing said light component, which has said first wavelength, said light component having been separated by said light splitting means from said light component, which has said second wavelength, an image of the condensed light component being thereby formed as a point image, PA0 xi) a second light receiving optical means for condensing said light component, which has said second wavelength, said light component having been separated by said light splitting means from said light component, which has said first wavelength, an image of the condensed light component being thereby formed as a point image, PA0 xii) a first photodetector for detecting said point image, which has been formed by said first light receiving optical means, and PA0 xiii) a second photodetector for detecting said point image, which has been formed by said second light receiving optical means.
An example of the confocal scanning microscope is disclosed in Japanese Unexamined Patent Publication No. 62(1987)-217218.
Also, a phase contrast microscope has heretofore been proposed, with which the phase information of a transparent object (i.e. a phase object) can be investigated. Basically, the phase contrast microscope comprises a phase plate, such as a quarter-wave plate, and a ring stop, which are located with a sample intervening therebetween. A light beam is irradiated to the sample. A phase lag is imparted to only either one of a light component of the light beam, which light component has been diffracted by the sample, and an undiffracted light component of the light beam, which undiffracted light component has passed through the sample or has been reflected by the sample. The diffracted light component and the undiffracted light component are then caused to interfere with each other, and the phase information of the sample is thereby converted into brightness and darkness.
The conventional confocal scanning microscope utilizes one of the following scanning mechanisms:
However, the scanning mechanism described in (1) has the problem in that the sample flies out of its correct position when it is scanned quickly. With scanning microscopes, samples of living organisms are often observed. If the quick scanning cannot be carried out during the observation of a sample of a living organism, subtle movements of the sample cannot be found. Also, a need exists widely for the real-time recording of images of various other samples. If the quick scanning cannot be carried out, such a requirement cannot be satisfied.
With the scanning mechanism described in (2), quick scanning can be achieved. However, the scanning mechanism has the drawback in that a light deflector, such as a galvanometer mirror or an acousto-optic light deflector (AOD), which is expensive must be used. Also, with the scanning mechanism described in (2), a light beam is deflected by a light deflector. As a result, the angle of incidence of the deflected light beam upon an objective lens of the light projecting optical means changes momentarily, and aberration is caused to occur. Therefore, the scanning mechanism described in (2) also has the problem in that it is difficult for the objective lens to be designed such that aberration can be eliminated. Particularly, in cases where an AOD is utilized, astigmatism occurs in the light beam radiated out of the AOD. Therefore, in such cases, a special correction lens must be used, and the optical means cannot be kept simple.
Also, various attempts have heretofore been made to constitute a phase contrast microscope as a confocal scanning type. However, the same problems as those described above occur with the conventional confocal scanning type of phase contrast microscope.